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At least 1,549 records · Page 86Linked to original sources
PubMed2003

An interface chip connection between capillary electrophoresis and thermal lens microscope.

A thermal lens microscope (TLM) detection of capillary electrophoresis (CE) utilizing microchip technology was developed. Fused-silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel in a microchip. The detection limit by TLM was estimated as 2.8 x 10(-7) absorbance by measuring pure water. The detection limit of derivatized amino acids determined by CE-TLM was estimated as 2.4 x 10(-8) M, which was 100 times lower than that of conventional absorbance detection.

Amino Acids↗
PubMed2004

The combination of flow injection with electrophoresis using capillaries and chips.

The combined flow injection capillary electrophoresis (FI-CE) system that integrates the essential favorable merits of FI and CE, can significantly expand the application scope of CE by exploring the various on-line sample pretreatments and preconcentration of FI. The principle behind this technique, some innovative designs of the split-flow interface, as well as novel applications to a variety of analytical problems, are reviewed and discussed. Some salient features and unique advantages of this technique are outlined.

Calibration↗
PubMed2004

Recent advances in polymeric monolithic stationary phases for electrochromatography in capillaries and chips.

This review article summarizes the advances made over the last two years in polymeric monoliths for capillary electrochromatography (CEC). It covers the scientific literature in the period extending form the second half of 2002 until the end of first half of 2004. Currently, there is an increasing interest in monolithic stationary phases in CEC as an alternative to particulate packed capillary columns due in major part to the simplicity of the in situ preparation of monolithic stationary phases and the availability of a wide chemistry for surface ligands, which allow for tailoring the chromatographic sorbent needed for solving a given separation problem(s). The various approaches, formats, and chemistries used for the preparation of monolithic stationary phases are described.

Acrylamides↗
PubMed2005

Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets.

Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA.

Cell Line, Tumor↗
PubMed2005

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution.

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.

Acrylic Resins↗
PubMed2005

Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips.

Separation of sodium dodecyl sulfate (SDS)-protein complexes is difficult on plastic microchips due to protein adsorption onto the wall. In this paper, we elucidated the reasons for the difficulties in separating SDS-protein complexes on plastic microchips, and we then demonstrated an effective method for separating proteins using polymethyl methacrylate (PMMA) microchips. Separation difficulties were found to be dependent on adsorption of SDS onto the hydrophobic surface of the channel, by which cathodic electroosmotic flow (EOF; reversed flow) was generated. Our developed method effectively utilized the reversed flow from this cathodic EOF as a driving force for sample proteins using permanently uncoated but dynamic SDS-coated PMMA microchips. High-speed (6 s) separation of proteins and peptides up to 116 kDa was successfully achieved using this system.

Adsorption↗
PubMed2005

Enzymatic sensitivity enhancement of biogenic monoamines on a chip.

Detection of biogenic monoamines in nanomolar concentrations is of great importance for probing the brain chemistry and for their analytics in biological fluids. The sensitivity enhancement of amperometric detection of neurotransmitters (NTs) and their metabolites after their electrophoretic separation on a microchip is presented and is based on coupled enzymatic reactions. The current response of the analyte is amplified by cyclic oxidation on a gold electrode mediated by reduced nicotinamide dinucleotide coenzyme and glucose oxidase enzyme present in the electrophoresis buffer. Using this approach, detection limits of about 10 nM for NTs and their metabolites can be reached.

Biogenic Monoamines↗
PubMed2005

Electrokinetic-driven microfluidic system in poly(dimethylsiloxane) for mass spectrometry detection integrating sample injection, capillary electrophoresis, and electrospray emitter on-chip.

A novel microsystem device in poly(dimethylsiloxane) (PDMS) for MS detection is presented. The microchip integrates sample injection, capillary electrophoretic separation, and electrospray emitter in a single substrate, and all modules are fabricated in the PDMS bulk material. The injection and separation flow is driven electrokinetically and the total amount of external equipment needed consists of a three-channel high-voltage power supply. The instant switching between sample injection and separation is performed through a series of low-cost relays, limiting the separation field strength to a maximum of 270 V/cm. We show that this set-up is sufficient to accomplish electrospray MS analysis and, to a moderate extent, microchip separation of standard peptides. A new method of instant in-channel oxidation makes it possible to overcome the problem of irreversibly bonded PDMS channels that have recovered their hydrophobic properties over time. The fast method turns the channel surfaces hydrophilic and less prone to nonspecific analyte adsorption, yielding better separation efficiencies and higher apparent peptide mobilities.

Dimethylpolysiloxanes↗
PubMed2006

A chip-based miniaturized format for protein-expression profiling: the exploitation of comprehensively produced antibodies.

Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology.

Animals↗
PubMed2006

Enantiomeric separation of underivatized small amines in conventional and on-chip capillary electrophoresis with contactless conductivity detection.

The determination of the enantiomers of small non-UV-absorbing amines which otherwise can only be achieved with difficulty was possible by using a combination of the chiral crown ether (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) and dimethyl-beta-CD as selectors in CE and contactless conductivity measurement for detection. Alkylamines without any other functional group, amino alcohols, species with ether or ester groups and with a cyclic moiety were investigated. The detection limits were found to be about 1.0 microM and the determination is possible up to at least 1.0 mM. The determination of enantiomeric ratios of up to 99.5:0.5 was also found feasible.

Amines↗
PubMed2007

Combination of flow injection with electrophoresis using capillaries and chips.

The technique of combined flow injection CE (FI-CE) integrates the essential favorable merits of FI and CE and can significantly expand the application of CE by utilizing the various on-line sample pretreatments and preconcentration of FI. The basic principles, instrumental developments, and applications of the FI-CE system from 2004 to 2006 are reviewed. The recent developments and applications of FI-CE are outlined.

Calibration↗
PubMed2006

Microelectronic DNA chip for hereditary hyperferritinemia cataract syndrome, a model for large-scale analysis of disorders of iron metabolism.

Hereditary hyperferritinemia cataract syndrome (HHCS) is caused by mutations in the regulatory iron responsive element (IRE) in the 5'UTR of the L-ferritin transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to a constitutive upregulation of the protein in tissue and serum. Twenty-nine mutations have been reported within the L-ferritin (FTL) IRE sequence, 21 of which were available to us. In addition, we included in this study three new mutations. Thus, we analyzed 24 mutations spanning over a DNA stretch of 48 nucleotides, including four deletions 2-29 nucleotides long and 20 substitutions, seven of which were conservative transversions. With this unique experimental model we developed a microchip diagnostic platform for identifying known molecular defects in the L-ferritin IRE structure with a microelectronic array approach, which we optimized after studying the effects of various parameters. The system enables electronic deposition of biotinylated amplicons to selected pads. Under optimized conditions, no cross-hybridization was found, even for mutations that affected the same or adjacent nucleotide positions. The same cartridge could be serially hybridized with all the 24 reporter probe sets, which allowed correct genotyping right up until the end of the analysis. Extensive validation on 200 samples in a blinded fashion gave total concordance of results. This pilot study represents a first step toward developing a diagnostic microchip for large-scale analyses for epidemiological studies and screening of mutations associated with iron disorders.

5' Untranslated Regions↗
PubMed2005

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients.

We applied a strategy that utilized a combination of systematic gene expression analysis with various tissues and immunological detection with sera from melanoma patients to identify melanoma antigens expressed preferentially in melanoma and melanocytes. We selected 101 genes by comparing cDNA profiles obtained by GeneChip analysis of a highly pigmented melanoma cell line, SKmel23, primary cultured melanocytes, HUVECs cultured endothelial cells, keratinocytes, liver and stomach. After the additional selection with criterion of high registered frequency of each cDNA in melanocyte-related cDNA libraries in the NCBI database, 15 genes including 12 known melanocyte specific genes were identified. One of the remaining 3 genes, FCRL/FREB, encoding a member of the Fc receptor family that was previously reported to express in germinal center B cells, was found to express preferentially in melanocytes and melanoma tissues by RT-PCR and Northern blot analysis. The FCRL/FREB protein was detected in the cytoplasm of melanoma cells by staining with the murine polyclonal antibody and by transfection with GFP-fused FCRL/FREB cDNA. The bacterial recombinant protein was recognized by serum IgG antibody obtained from some patients with melanoma. These results suggest that FCRL/FREB may function in melanocytes and melanoma and may be useful for development of diagnostic methods for various pigment disorders and immunotherapy of melanoma.

Animals↗
PubMed2004

Proteomic profiling of platelet proteins by trypsin immobilized self-assembled monolayers digestion chip and protein identification using electrospray ionization tandem mass spectrometry.

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. Recently, the bonding of enzyme to SAMs of alkanethiols onto Au electrode surfaces was exploited to produce a bio-sensing system. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agent. Experimental results have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s core levels at 286.3 and 286.5 eV (C with N), 288.1 eV (amide bond), and 289.3 eV (carboxyl) illustrate the immobilization of trypsin. These data were also in good agreement with Fourier-Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks valued at 1659.4 cm(-1) (amide I) and 1546.6 cm(-1) (amide II). Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) observations, analytical results have demonstrated the platelet proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, platelet proteins were digested on the trypsin-immobilized SAMs surface, which shows the enzyme digestion ability of the immobilized trypsin. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.

Blood Platelets↗
PubMed2004

Is hydroxyapatite cement an alternative for allograft bone chips in bone grafting procedures? A mechanical and histological study in a rabbit cancellous bone defect model.

To evaluate in vivo performance of hydroxyapatite cement (HAC) as a porous bone graft substitute, HAC was mixed (1:1 ratio) with either porous calcium-phosphate granules (80% tricalcium phosphate, 20% hydroxyapatite) or defatted morsellized cancellous bone (MCB) allograft and implanted bilaterally in cylindrical drill holes in distal femurs of rabbits. Groups with empty defects and impacted MCB were used for reference. After 8 weeks, one femur from each pair was examined histologically. All contralateral specimens and Time-0 specimens were used for mechanical indentation tests. Histology showed that some empty defects were filled with newly formed osteopenic bone after 8 weeks. The impacted MCB showed remodeling into new vital bone. Incorporation of the HAC/MCB composite was incomplete, whereas minimal new bone ingrowth was found in the HAC/granule composites. Though not different from each other, both composites were significantly stronger than empty defects, incorporated impacted MCB, and intact cancellous bone. At Time 0, the mechanical behavior of impacted MCB was similar to both HAC composites. In conclusion, composites of HAC and porous biomaterials can maintain relatively high strength over 8 weeks in vivo, but their incorporation into a new bony structure is slower than impacted MCB. The HAC/MCB composite showed favorable incorporation behavior.

Animals↗
PubMed2007

Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter.

Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.

Animals↗
PubMed1998

Y2K, embedded chips, casualty hazards and due diligence in healthcare risk management.

Y2K raises challenges for healthcare risk managers that go beyond information technology issues. This article explains that (1) too little public attention is being paid to equipment which may well have Y2K faults and (2) few standards have been articulated for dealing with problems. Healthcare risk managers therefore must return to basic due diligence principles and develop their own standards and protocols. The article explains how to do due diligence and outlines suggested steps for dealing with the non-information technology side of compliance due diligence.

Budgets↗
PubMed2004

Improving resolution for channel-format chip-based electrophoresis with electrochemical array detection.

Resolution in channel electrophoresis has been improved by means of the addition of a surfactant to the running buffer and minimization of the channel internal height and sampling capillary internal diameter. Micellar electrokinetic channel chromatography with electrochemical detection has been applied to the separation of several cationic catecholamines and has been used to continuously monitor a dynamic system of dopamine, norepinephrine, and epinephrine. Resolution was also enhanced by coupling small internal-diameter (5 microm) sampling capillaries with sub-micrometer internal-height separation channels. The improvements in resolution offered by these methods will extend the applicability of channel electrophoresis with electrochemical detection to more complex samples while permitting sample volumes in the nL range to be probed.

Buffers↗